Segmental flexibility of bispecific T-cell engagers regulates the dynamics of immune synapse formation

Abstract

AbstractBispecific T-cell engagers (TcEs) are antibody-based immunotherapeutic drugs that specifically direct lymphocyte effector functions against tumors. TcEs have one arm with affinity for an activating immunoreceptor connected through a flexible hinge to a distinct arm with affinity for a tumor antigen to achieve tumor killing by cytotoxic immune cells. Understanding the structure-function relationships between TcE architecture, immunological synapse formation and function could accelerate design of new TcE-based cancer therapies. Here, we engineer and systematically characterize TcE formats with antigen binding antibody fragments or single chain variable fragments linked together incisthrough immunoglobulin G1 hinge or intransacross the antibody constant fragment. The TcEs were tested in CD8+T-cell killing of Her2+breast cancer cells and evaluated by high-content imaging of immunological synapse formation on a supported lipid bilayer (SLB) platform. We find thatcisTcEs perform better than atransTcE for T-cell mediated killing. Quantification of synapse formation dynamics revealed that all threecisTcEs tested, created close contacts of < 16 nm leading to rapid synapse formation and integrin activation. In contrast, thetransTcE formed close contacts averaging ≥ 16 nm and formed synapses more slowly with weaker integrin activation. We conclude that segmental flexibility is important for TcE function, but adding additional degrees of freedom throughtransformats has a cost for killing efficiency that may be explained by failure of close contact formation and integrin activation.